HIGHER EDUCATION COMMISSION
NATIONAL RESEARCH PROGRAM OF UNIVERSITIES
FINAL REPORT
Reporting period from: June, 2003
to: June, 2006
HIGHER EDUCATION COMMISSION
NATIONAL RESEARCH PROGRAM OF UNIVERSITIES
FINAL REPORT
PART: 1
GENERAL INFORMATION
TITLE OF PROJECT:
Screening for Vision and Hearing Impairment in Rural Sindh and Balochistan
REPORTING PERIOD FROM : June, 2003
PRINCIPAL INVESTIGATOR’S NAME:
To: June, 2006
Sheikh Riazuddin
PRINCIPAL INVESTIGATOR’S INSTITUTE MAIL ADDRESS:
Centre of Excellence in Molecular Biology, University of the Punjab, 87- West Canal Bank
Road, Thokar Niaz Baig, Lahore-53700, Pakistan.
TELEPHONE:
E-MAIL:
92-42-5421235
FAX:
92-42-5164155
riaz@lhr.comsats.net.pk
ABSTRACT:
Sixty large consanguineous families comprising four or more affected siblings were
enrolled as part of the studies to Screen for Vision and Hearing Impairment in Rural Sindh and
Balochistan. The levels of hearing impairment was determined by audiometric analysis and
vision impairment was determined by fundoscopy and electroretinography (ERG).
Blood samples were collected from affected as well as non-affected siblings and living
parents and grand parents. DNA was isolated and processed for linkage analysis for the presence of
known DFNB loci and RP loci. Twenty three families were found linked to DFNB1, B2, B3, B4,
B7/11, B9, B12, B21, B23, B29 & B37. Ten families were found linked to PDE6A, TULP1, RPE65,
CNGB1/BBS2 & CNGA1. Sequencing of the four PDE6A linked families identified three different
mutations while in the fourth family linked to the same region, no disease causing mutation was
identified in PDE6A gene. Genome wide scan was carried out on selected six unlinked vision
impaired families in search for new Retinitis pigmentosa (RP) loci/genes. A new recessive locus
mapped to a 14.21 cM (21.19 Mb) region on chromosome 8q11 was identified in three families.
Sequencing of the coding exons of RP1 known to cause dominant RP shows mutations in all 3
families. Another new recessive locus RP32 was mapped on chromosome 1 P13.3 – P21.2 that
causes severe autosomal recessive RP. Among the DFNB linked families, three DFNB1 families
upon sequencing revealed two mutations in the coding exon of GJB2 gene. Forty nine exons of
MYO7A gene were sequenced in the five families linked to DFNB2/USH1B and three mutations
were identified. Sequencing of 21 exons of SLC26A4 gene in the five families linked to
DFNB4/PDS, identified mutations in exons 3, 6 & 19.
Genome wide scan was carried out on selected four unlinked hearing impaired families in
search for new deafness loci/genes. A new locus DFNB48 mapped to chromosome 15 q 23–25.1
was identified in two families from Sindh province (Ahmad, et al., 2005). The DFNB48 locus
acronym
was
provided
by
Human
Genome
Organization
Committee
(HUGO):
http://www.gene.ucl.ac.uk/hugo. In one family form Balochistan another new locus mapped to
chromosome 6p21.1-p22.3 was identified with locus acronym DFNB67. Sequencing of the
coding exons of TMHS shows mutation segregating with the hearing phenotype (Shabbir et al.,
2006). Normal allele frequencies for the markers in the region of DFNB48 and DFNB67 were
calculated by genotyping 90 normal random samples collected from the same province.
The above results have contributed to our understanding of the problem of the prevalence/
role of mutations that cause hearing/vision impairment in different ethnicities in Sindh and
Balochistan.
PART: 2
BACKGROUND / METHODOLOGY
BACKGROUND INFORAMTION:
Retinitis pigmentosa (RP) is a group of inherited diseases that affect the retina. The
disease is characterized by a gradual break down and degeneration of photoreceptor cells (Rods
and Cones) that results in a progressive loss of vision. The rate of progression and degree of
visual loss are variable. The symptoms of RP include night blindness, loss of peripheral vision
and ability to discriminate colors.
RP is classified as non syndromic and syndromic. Non syndromic RP may be familial
(Multiplex) or isolated (Simplex). Mode of inheritance is Autosomal dominant, Autosomal
recessive, X-linked, and Mitochondrial. To date 47 non syndromic loci have been localized of
which 37 genes are known.
The spectrum of hereditary deafness is broad and ranges from simple deafness without
other clinically recognizable abnormalities (non-syndromic) to genetically determined syndrome
of a more pleiotropic nature. Syndromic form with anomalies of the eye (Usher Syndrome) is the
most common syndrome associated with hearing impairment (Bergstorm, et al., 1971).
A number of mutated genes have been identified that are responsible for syndromic
deafness, associated with vision impairment and it is believed that efforts for search for new
genes / loci is not at all completely exhausted. Localization of genes responsible for recessive
hearing / vision loss is often difficult because of the extreme heterogeneity of these genetic
disorders and small family sizes in many countries (petit, 1996). Besides locus heterogeneity,
allelic heterogeneity is common. The allelic heterogeneity is sometimes associated with clinical
heterogeneity, with non-syndromic hearing / vision impairment being caused by mutations
within the same gene. Furthermore, different mutations within the same gene result in a variety
of phenotypes e.g mutation of MYO 7A can cause Usher syndrome USH1B, dominant
DFNA11and recessively inherited non-syndromic deafness DFNB2 (Liu, et al 1997, Weil, et al.,
1997). In a background of the above, it was proposed to expand the scope to extend the ongoing
studies on hearing / vision impairment to scan different ethnicities in interior Sindh and
Balochistan. A single inbred family may have several deaf / vision impaired individuals, which
will be helpful in linkage analysis as well as cloning of the newly discovered genes. The over all
objective of the proposed studies is to obtain information on the prevalence of mutations in genes
as well as new loci involved in hearing / vision impairment.
WORK PROGRESS/ RESULTS
Sixty large consanguineous families (35 hearing and 25 vision impaired) having four or
more affected individuals were enrolled in this programme. Detailed medical history with any
observable clinical symptom was recorded.
Audiograms of selected deaf individuals, their
parents and siblings were conducted. An ophthalmologist examined those individuals with a
history of vision impairment for fundoscopy and field analysis. In certain selected cases, the
affected individuals were transported to CEMB to confirm the diagnosis of retinitis pigmentosa
by Electro-retinography (ERG). Blood samples were collected and genomic DNA was extracted
by standard methods. Genomic DNA was analyzed by genotyping for known DFNB loci.
Twenty three hearing impaired pedigrees were identified as linked to reported loci (Table-1).
TABLE – 1 :
DEAFNESS LOCI
DFNB 1
DFNB 2
DFNB 3
DFNB 4
DFNB 7/11
DFNB 9
DFNB 12
DFNB 21
DFNB 23
DFNB 29
DFNB 37
Total
NO. OF LINKED
PEDIGREES
3
5
2
5
1
1
1
1
1
1
2
23
Sequencing for GJB2 (DFNB1)
It has been reported that hearing impairment that cosegregates with DFNB1, is caused by
mutations in GJB2 gene (Guilford et al. 1994). GJB2 gene is located on chromosome13 at
position 13q11and has two exons. Exon 1 encodes the 5’ untranslated region and exon 2
contains the entire open reading frame (680 bp) encoding 208 amino acid long protein product
called connexin26 (Cx26). Coding exon 2 was sequenced in the three linked families and the
results are shown below.
Exon
2
2
Mutation
W77X
W24X
Sequencing for MYO7A (DFNB2/USH1B)
DFNB2/Usher 1B is located on chromosome 11 at 11q13.5 and includes 49 exons
(Weil, D. et al.1997). . All the 49 exons in five linked families were sequenced. The following
three mutations were identified:
Exon
Mutation
7
G214R
21
A826T
28
E1170K
Sequencing for SLC26A4 (DFNB4/PDS)
SLC26A4 gene lies on chromosome 7 at 7q31 (Baldwin et al. 1995). SLC26A4 has 21
exons. All exons in the five linked families were sequenced and three mutations were identified.
Exon
3
6
19
Mutatiom
S90L
V239D
K715N
Genome wide scan was carried out on selected four unlinked hearing impaired families in
search for new deafness loci/genes. A new locus DFNB48 mapped to chromosome 15 q 23–25.1
was identified in two families from Sindh province (Ahmad, et al., 2005). The DFNB48 locus
acronym was provided by Human Genome Organization Committee (HUGO):
http://www.gene.ucl.ac.uk/hugo. In one family form Balochistan another new locus mapped to
chromosome 6p21.1-p22.3 was identified with locus acronym DFNB67. Sequencing of the
coding exons of TMHS shows mutation segregating with the hearing phenotype (Shabbir et al.,
2006). Normal allele frequencies for the markers in the region of DFNB48 and DFNB67 were
calculated by genotyping 90 normal random samples collected from the same province.
When genomic DNA of vision impaired families were analyzed by genotyping for known
RP loci, the ten families were found as linked to reported loci (Table-2)
TABLE – 2 :
RP LOCI
PDE 6A
TULP1
RPE65
CNGB1/BBS2
CNGA1
Total
NO. OF
PEDIGREES
4
2
2
1
1
10
Genome wide scan was performed on selected six pedigrees unlinked to the reported RP
loci. In one family linkage was detected on chromosome 1p13.3-p21.2 defining a novel locus
RP32. This new locus resides on chromosome 1 between D1S2896 and D1S457 & causes severe
autosomal recessive RP. Another linkage was detected on chromosome 8q11 in one family
defining a novel locus of Retinitis pigmentosa. In order to confirm this linkage and fine map the
genetic interval, additional families present in CEMB repository were screened and two more
families were linked to the same region. RP in all 3 families mapped to a 14.21 cM (21.19 Mb)
region on chromosome 8q11 flanked by D8S532 and D8S260. This region harbors RP1, which
is known to cause autosomal dominant retinitis pigmentosa. Sequencing of the coding exons of
RP1 shows mutations in all 3 families: two single base deletions, c.7470delA and c.8168delA
resulting in a frame shift and a 4bp insertion c4377-4378insTGAA all causing premature
termination of the protein.
Rp in four families maps to a 13.85 cM (14.87 Mb) region on chromosome 5q31-33
flanked by D5S2090 and D5S422. This region harbors the PDE6A gene, which is known to
cause arRP. Sequencing of PDE6A shows a single base pair change c.889→ T, a single base pair
insertion c.2218→ 2219 ins T and a single base pair substitution in the splice accepter site IVS
10-2 A→G in three families. In the fourth family no disease causing mutation was identified in
the PDE6A gene.
OBJECTIVES / ACHIEVEMENTS
Approved objectives





Completed
Search for families in rural Sindh & Balochistan
with 3 or more hearing and vision impaired individuals.
100%
Collection of blood samples from the affected
and close relatives in the pedigree and DNA
Extraction from affected and normal individuals.
100%
Clinical tests, Audiograms, Fundoscopy,
Electroretinography (ERG) to identify the type of deafness
and vision impairment in the family.
100%
Genotyping for screening the reported
loci and search for new loci.
100%
Mutation screening.
100%
METHODOLOGY
Blood and Buccal Swab Collection
Blood samples (10cc) were collected from parents, affected children and normal siblings
of each nuclear family along with all the available elder members of the Pedigree. EDTA was
used as an anticoagulant. Blood was kept at 4OC before processing for DNA extraction. In case
of elderly or small children buccal swabs (Epicentre Technologies WI, Medical Package
Corporation, CA, USA) were used to collect buccal epithelial cells.
DNA Extraction
Blood samples were processed to extract DNA by a non-organic method (Grimberg et al.,
1989). DNA concentrations were determined by spectrophotometer. DNA from buccal swabs
was extracted according to the instructions of manufactures Epicentre Technologies WI, Medical
Package Corporation, CA, USA.
Linkage analysis for known DFNB & RP loci
Efforts were made to ascertain the families, which fall into the already known linkage
region. For this purpose, reported DNFB & RP markers were used. The microsatellite markers
were amplified by polymerase chain reaction, using genomic DNA as a template. These markers
are listed in genome data base (GDB) and only fluorescent markers were used. The PCR product
of the markers were pooled in such a way that the marker size and their dye do not overlap. The
PCR products were analyzed on the ABI 3100 genetic analyzer and alleles were assigned using
Genescan and Genotype Software. Scans showing various allele patterns for the microsatellitc
markers, were labeled and placed on the corresponding pedigrees to find out whether the marker
cosegregates with the disease gene. In this way the families that link to the known loci were
ascertained and those that do not link to any of the known locus were separated.
Mutational Analysis
BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer/Applied
Biosystems) was used to sequence the PCR products (RP1). The thermal cycling was done in
GeneAmp PCR system 9700 & 2700 (Perkin Elmer). 10μl deionized formamide (Perkin Elmer)
was used to suspend the pellet and loaded on ABI 3100 genetic analyzer/sequencer according to
the manufacturer’s instructions. Any change in the DNA sequence was confirmed by sequencing
both sense and antisense strands. Once a mutation is confirmed, samples from all available
affected individuals and their normal siblings were also sequenced to confirm that the change in
the DNA sequence is segregating with affected phenotype.
Genome wide scan
Genome-wide search was undertaken using 358 fluorescently labeled microsatellite
markers at an average spacing of 10 cM (ABI, Prism 1, Linkage mapping set 2.5, Applied
Biosystems). 2 µl of the PCR product along with 11.8 µl of formamide and 0.2 µl LIZ size
standard (Applied Biosystems) was pooled for genotyping. Amplified products were resolved by
capillary electrophoresis using an ABI Prism 3100 Genetic Analyzer. The alleles were assigned
using Genescan and Genotyper software (Applied Biosystems).
Lod score calculation
For linkage analysis, the Marshfield genetic map was used for marker order and map
distances. Lod scores were calculated by using MLINK of the FASTLINK computer package.
Disease was coded as fully penetrant and disease allele frequency was set at 0.001. Meiotic
recombination frequencies were assumed to be equal for males and females.
PUBLICATIONS
1.
Ahmad J., Khan S N., Khan S. Y., Ramzan K., Riazuddin S., Ahmed Z M., Wilcox E
R., Friedman T B., Riazuddin S. 2005. DFNB48, a new Nonsyndromic recessive
deafness locus maps to chromosome 15q23-q25.1. Hum Genet. 116:407-412.
2.
S.Amer Riazuddin, Fareeha Zulfiqar, Qingjiong Zhang, Yuri V.Sergeev
Zaheeruddin.A.Qazi, Tayyab Hussnain, Raphael Caruso, Sheikh Riazuddin, Paul. A.
Sieving, J. Fielding Hejtmancik. 2005. Autosomal Recessive Retinitis pigmentosa is
associated with mutations in RP1 in three consanguineous Pakistani families.
Investigative Ophthalmology & Visual Science (IOVS). 46(7): 2264-2270.
3.
Qingjiong Zhang, Fareeha Zulfiqar, Xueshan Xiao, S. Amer Riazuddin, Muhammad
Farooq Sabar, Raphael Caruso,Paul A. Sieving, Sheikh Riazuddin, J. Fielding
Hejtmancik 2005. Severe recessive retinitis pigmentosa maps to chromosome 1p13.3p21.2 between D1S2896 and D1S457 but outside ABCA4. Hum Genetics. 28:1-10.
4.
Shabbir M I, Ahmed Z M, Khan S Y, Riazuddin S, Waryah A M,Khan S N, Camps
R D, Ghosh M, Kabra M, Belyantseva I A, Friedman T B, Riazuddin S. 2006.
Mutations of human TMHS cause recessively inherited nonsyndromic hearing loss. J
Med Genet. 43(8): 634-40.
5.
S.Amer Riazuddin, Fareeha Zulfiqar, Qingjiong Zhang, Wenliang Yao, Shouling Li,
Xiaodong Jiao, Amber Shahzadi, Muhammad Amer, Muhammad Iqbal,Tayyab
Husnain, Paul Sieving, Sheikh Riazuddin, J. Fieldingg Hejmancik. 2006 Mutations in
the gene encoding the α–subunit of rod & phosphodiesterase in Consanguineous
Pakistani families. Molecular vision 12:1283-1291.
BACKGROUND / METHODOLOGY continued
REFERENCES

Ahmad J., Khan S N., Khan S. Y., Ramzan K., Riazuddin S., Ahmed Z M., Wilcox E R.,
Friedman T B., Riazuddin S. 2005. DFNB48, a new Nonsyndromic recessive deafness locus
maps to chromosome 15q23-q25.1. Hum Genet. 116:407-412.

Bergstorm, L et al., (1971): A high risk registry to find congenital deaness. Otolaryngol Clin
North Am; 4: 369 -99.

Grimberg, J. et al (1989). A simple and efficient non organic procedure for the isoaltion of
genomic DNA from blood. Nucleic Acid. Res. 17: 8390-8391.

Liu, et al. (1997). Muations in the Myosin VIIa gene cause non-syndromic recessive
deafness, Nat Genet:16:188-190.

Petit, (1996). Genes responsible for hereditary deafness - symphony of a thousand Nat Genet
14: 385-9.

Shabbir M I, Ahmed Z M, Khan S Y, Riazuddin S, Waryah A M,
Khan S N, Camps R
D, Ghosh M, Kabra M, Belyantseva I A, Friedman T B, Riazuddin S. 2006. Mutations of
human TMHS cause
recessively inherited nonsyndromic hearing loss. J. Med. Genet.
Electronically published February 2006.

Weil, D. et al. (1997). The autosomal recessive isolated deafness DFNB2 and the Usher 1B
are allelic defects of the myo VIIa gene. Nat Genet 16: 191-3.
SUMMARY
Sixty large consanguineous families comprising four or more affected siblings
were enrolled as part of the studies to Screen for Vision and Hearing Impairment
in Rural Sindh and Balochistan. The level of hearing impairment was measured
by pure tone audiometry and vision impairment was determined by fundoscopy
and electroretinography (ERG).
Blood samples were collected from affected as well as un-affected siblings
and living parents and grand parents. DNA was isolated and processed for linkage
analysis for the presence of known loci. Twenty three families were found linked to
known deafness loci and ten families were found linked to blindness loci. DNA
sequencing studies identified different pathological mutations in Sindhi or Balochi
people (Riazuddin et al., 2006). Genome wide scan was carried out on selected six
unlinked vision impaired families in search for new Retinitis pigmentosa (RP)
loci/genes. A new recessive locus mapped to a 14.21 cM (21.19 Mb) region on
chromosome 8q11 was identified in three families. Sequencing of the coding exons
of RP1 known to cause dominant RP shows mutations in all 3 families (Riazuddin et
al., 2005). Another new recessive locus RP32 was mapped on chromosome 1
P13.3 – P21.2 that causes severe autosomal recessive vision impairment.
Genome wide scan was carried out on selected four unlinked hearing
impaired families in search for new deafness loci/genes. A new locus DFNB48
mapped to chromosome 15 q 23–25.1 was identified in two families from Sindh
province (Ahmad, et al., 2005). The DFNB48 locus acronym was provided by
Human Genome Organization Committee (HUGO): http://www.gene.ucl.ac.uk/hugo.
In one family form Balochistan another new locus mapped to chromosome
6p21.1-p22.3 was identified with locus acronym DFNB67.
The results of the above studies have a strong bearing on our understanding of
the processes of hearing and vision and the prevalence/ role of mutations that
cause hearing/vision impairment in different ethnicities in Sindh and Balochistan.
Information on carrier status was provided to the affected families on request for
genetic counseling. This information will inevitably help to reduce the incidence of
deafness and blindness resulting from high consanguinity in these families. The
results of these studies have been published in five peer reviewed international
journals.